2) Multifactorial Genetics and the Genetic analysis of type 1 diabetes in the mouse.

3) Stem Cells and the role of the X-linked gene Nap1l2 in the control of neuronal stem cell proliferation

One of the major research interests of the Mouse Molecular Genetics Unit is X chromosome inactivation.This is a complex biological process which depends on the presence of the X-inactivation centre (Xic) and which results in the inactivation of one of the two X chromosomes present in the female cell. The process involves both the sensing and counting of the number of X chromosomes in the cell in relation to the autosomal complement and a choice process concerning the X to inactivate. Whilst several genetic elements including the genetically identified locus Xce (X-controlling element) seem to be able to affect the choice of which X chromosome will be chosen to be inactivated, little is known about the factors involved in the counting process. The Xic increasingly appears as a complex control region with multiple elements feeding into the regulation and interactions with the Xist (X-inactive specific transcript) gene which codes for a large non-coding RNA which plays a critical role in the initiation of the inactivation process.

A major part of the work in the laboratory is concerned with the functional identification and analysis of the various components of the Xic involved in the counting and choice mechanisms. Our double approach strategy exploits on the one hand, the creation of novel mutations by targeted mutagenesis using the cre-lox system, and on the other the complementation of pre-existing engineered deletions. Use of the complementation or so-called add-back strategy has allowed us to localise the counting element(s) to a 20kb candidate region. Other parts of this 3' region known to control both the expression of Xist and Tsix antisense have now been shown to influence both the retention of Xist RNA at its chromosomal site of transcription and the chromatin state', or the openess of the chromatin at the Xist promoter region.

A better understanding of some of the processes involved in the initiation of X-inactivation is likely to depend on combining such genetic and cell biology approaches with a detailed analysis of chromatin structure. Several different approaches to such an analysis including ChIP (Chromatin Immunoprecipitation) have been established in the laboratory. ChIP studies have recently proved of great importance in characterising the role played by histone H3 methylation in the earliest stages of X-inactivation and have allowed us to show define a region 5' to Xist which may act as a nucleation centre allowing the inactivation to spread from the Xic over the whole X chromosome. Detailed analysis of histone modifications occurring in this region have been undertaken and a correlative analysis of such epigenetic changes with the underlying genomic sequence initiated. These studies have allowed the definition of a sub-region which appears of particular interest and which is now being analysed genetically.

Genetic factors involved in the X-inactivation process lying outside of the Xic and in all probability elsewhere than on the X chromosome, are being looked for by a complementary approach based on a comparative transcriptome analysis of 6.5 dpc female and male embryos using SAGE (Serial Analysis of Gene Expression) analysis. Tags showing differential expression have been subject to serial screens aimed at confirming their status potential prior to extended functional analysis. The latter is based on inhibition of gene function in vitro by RNAi and observation of the resulting status of the inactivation process and the inactive X chromosome.

Genome Research and Mouse Disease Models

The Genetic analysis of type 1 diabetes

Another of the Unit's interest concerns the study of mouse phenotypes under multifactorial and polygenic control. We have taken as our prototype type 1 diabetes or insulin- dependent diabetes for which the NOD mouse represents an interesting model. Our studies are aimed at defining the genetic factors (Idd) implicated in this pathology which is known to depend on a complex interaction between environmental and genetic factors. Our studies are concentrated on the characterisation of Idd loci controlling diabetes susceptibility/resistance locating to the distal part of mouse chromosome 6. The past year has seen major advances in the definition and localisation of three loci Idd6, Idd19 and Idd20 lying in the distal part of mouse chromosome 6.

The continued refining of the candidate regions for these loci by establishment of congenic mouse strains has allowed candidate gene approaches to be undertaken to defining and characterising the genes responsible for these traits and in particular for Idd6. Four new congenic lines have been exploited during the past twelve months. The immunological characterisation of our congenic strains has allowed the definition of the site of action/tissue involved in resistance to diabetes onset conferred by the Idd6 locus. Varied approaches including transcriptional profiling have allowed candidate genes for the Idd6 locus to be restrained to less than half a dozen which are now being analysed functionally using RNAi based approaches.

Stem Cells and the role of the X-linked gene Nap1l2

A second disease subject to both environmental and complex genetic control is spina bifida. In the mouse spina bifida can be induced by mutation in theX-linked Nap1l2 gene. Mutations in this gene are associated with embryonic lethality, spina bifida and exencephaly linked to a massive overproliferation of neuronal cells. We have now been able to obtain conditional mutations in the Nap1l2 gene and these are being exploited to explore its role in the adult. The recent establishment of a highly efficient system for differentiating ES cells into neural stem cells and cells of the neuronal ligneage has opened up interesting perspective for understanding the role and mechanism of action of this gene in tissue specific cell cycle regulation including neuronal stem cells which are the site of its action.